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Screening and Cloning of RAPD Markers from the W Chromosome of Silkworm, Bombyx mori L.
Chen, Keping,Zhang, Chunxia,Yao, Qin,Xu, Qinggang,Tang, Xudong Korean Society of Sericultural Science 2004 International Journal of Industrial Entomology Vol.8 No.2
Silkworms sex determination drew high attention from researchers. Sex chromosomes on the silkworm are of ZW type for females and ZZ type for males. Chromosome W plays an important role in sex determination. Although several molecular linkage maps have been constructed for silkworm, very few markers are discovered on the W chromosome. In order to look for molecular markers and to further locate the Fern gene on chromosome W, we used genomic DNA from both female and male larvae of a silkworm strain named 937 as PCR templates for RAPD amplification with 200 arbitrary 10-mer primers. The amplification results showed three female-specific bands, namely ${OPG-07_496}, {OPC-15_1,660} and {OPE-18_1,279}$. Further verification, however, revealed no band from OPG-07 and OPC-15 in either sex in the strain 798, but OPE-18 provided female-specific band in the strains Suluan7 and C108, and absent in both males and strain 798. This indicates that the bands from ${OPG-07_496} and {OPC-15_1,660}$ are probably female-specific in strain 937, and the band from OPE-18 was probably amplified from a common segment shared by most strains. The genomic DNAs from OPG-07 and OPC-15 were cloned and sequenced. Sequence analysis showed that the DNAs from OPG-07 and OPC-15 have high identities with the retrotransposable elements, and DNA from OPC-15 contains a portion of sequence which probably encodes an eukaryotic translation initiation factor 4E binding protein (eIF4EBP).
Screening of RAPD Markers for Fluoride Resistance in Bombyx mori L.
Chen, Keping,Yao, Qin,Li, Muwang,Wang, ong Korean Society of Sericultural Science 2003 International Journal of Industrial Entomology Vol.7 No.1
NF733xin, the near allele line was obtained by means of crossing and backcrossing the silkworm race T6, which contained fluoride resistance major gene, to race 733xin, which was highly susceptible to fluoride toxicity. Two hundred RAPD random primers were used in the RAPD analysis of these 3 strains. Two molecular markers, OPB-08850 and OPB-10917, were obtained. OPB-10917 was used to detect the backcross generations. It was found that all the fluoride resistant individuals in each backcross generation had the same special band. These results proved that this marker was reliable.
Chen, Keping,Yao, Qin,Wang, Yong,Cheng, Jialin Korean Society of Sericultural Science 2003 International Journal of Industrial Entomology Vol.7 No.1
The nuclear polyhedrosis virus (NPV) resistance of silkworm is controlled by a pair of dominant genes on autosome and micro-effect modificator genes on sex chromosome Z and has the phenomenon of patroclinal inheritance. Based on its hereditary characteristics, methods of preparing near isogenic lines and their $F_2$ populations for screening molecular markers were designed.
Lu, Peng,Chen, Keping,Yao, Qin,Yang, Hua-Jun Korean Society of Sericultural Science 2007 International Journal of Industrial Entomology Vol.15 No.2
As the genome of B.mori is available in GenBank and the EST database of B.mori is expanding, identification of novel genes of B.mori is conceivable by data-mining techniques. We used the in silico cloning method to get the vacuolar-type $H^+-ATP$ synthetase (V-ATPase) c subunit (16 kDa proteolipid subunit) gene of B.mori and analysed with bioinformatics tools. The result was confirmed by RT-PCR and sequencing. The V-ATPase c subunit cDNA contains a 468 bp ORF. The ORF encoded a 155-residue protein that showed extensive homology with V-ATPase c subunits from other 15 species and contained four membrane-spanning helices. Tissue expression pattern analysis revealed that V-ATPase c expressed strongly in Malpighian tubules, not in fat body. This gene has been registered in GenBank under the accession number EU082222.
Rapid Expression of Bm46 in Bombyx mori Cell Lines, Larvae and Pupae
Wang, Haiyan,Chen, Keping,Guo, Zhongjian,Yao, Qin,Wang, Qiang,Mu, Runhong Korean Society of Sericultural Science 2007 International Journal of Industrial Entomology Vol.15 No.1
In this study, ORF 46 of Bombyx mod nucleopolyhedrovirus(Bm46) fused with EGFP was expressed in Bombyx mod cell lines, larvae and pupae by BmNPV Bacmid system. Bm46 and EGFP were cloned into donor plasmid pFastBacHTb, which was transformed to competent DH10B cells containing helper and BmNPV bacmid by site-specific transposition. Recombinant bacmid was used to transfected BmN-4 cells to produce the recombinant baculovirus vBm-Bm46-EGFP. Recombination virus was injected into silkworm larvae and pupae. The expression of the fusion protein was monitored by examining green fluorescence using a fluorescent microscope. Intense fluorescence in cells and silkworm was observed at 4 days post-infection, indicating the Bm46-EGFP fusion gene was expressed successfully.
Cloning and Characterization of hydroxypyruvate isomerase (EC 5.3.1.22) gene in silkworm Bombyx mori
Lv, HongGang,Chen, KePing,Yao, Qin,Wang, Lin Korean Society of Sericultural Science 2008 International Journal of Industrial Entomology Vol.17 No.2
The sequence of hydroxypyruvate isomerase gene was obtained in NCBI. In this study, the hydroxypyruvate isomerase gene of Bombyx.mori was identified and annotated with bioinformatics tools. The result was confirmed by RT-PCR, prokaryotic expression, mass spectrographic analysis and sub-cellular localization. The hydroxypyruvate isomerase cDNA comtains a 783bp ORF, and has 4 exons. The deduced protein has 260 amino acid residues with the predicted molecular weight of 29169.30 Da, isoelectric point of 6.10, and contains conserved PRK09997 and Hfi domains. The hydroxypyruvate isomerases of Nasonia vitripennis and Bombyx mori have a high homology. Through RTPCR analysis, we found that this transcript was present in testis, ovary, blood-lymph, fat body, midgut, silk gland and tuba Malpighii. This protein was located in cytoplasm through immunohistochemistry. We submitted the cloned gene under the accession number EU344910. The enzyme has been classified under accession number EC 5.3.1.22.
Identification and Characterization of Bombyx mori LDH Gene through Bioinformatics Approaches
Zhu, Minfeng,Chen, Keping,Yao, Qin Korean Society of Sericultural Science 2007 International Journal of Industrial Entomology Vol.15 No.2
Lactate dehydrogenase (LDH) is a ubiquitous enzyme that plays a significant role in the clinical diagnosis of pathologic processes. Discovery of the LDH (BmLDH) gene in B. mori may shed light on its role in the biology of Lepidoptera species, and afford further understanding of the function of the enzyme. In this study, we used the bioinformatics tools to identify LDH gene in B. mori. Sequence analysis showed that BmLDH cDNA contains a 996 bp open reading frame, encoding 331 AA proteins, with seven introns. Compared with hHLDH (human heart LDH), BmLDH contained the same key active sites. Domain search and protein fold recognition analyses provide compelling evidences that the deduced protein is a LDH. Using the computer program MEGA3, we conducted a search for homologs of BmLDH among many eukaryotic species and confirmed that the BmLDH was conserved in all organisms investigated. This gene has been registered in GenBank under the accession number EU000385.
RNA Interference to Prevent Bombyx mori Nuclear Polyhedrosis Virus Infection in Vivo
Hu Zhigang,Chen Keping,Gao Lu,Yao Qin Korean Society of Sericultural Science 2006 International Journal of Industrial Entomology Vol.12 No.1
RNA interference has been used as a powerful tool in preventing virus proliferation in many species. In this study, we injected the dsRNA in vitro transcripts into Bombyx mori to investigate the resistance to B. mori nuclear polyhedrosis virus (BmNPV). Through vivisectional observation and real-time quantities PCR analysis, we found that these dsRNA can prevent the BmNPV to a certain extent, and delay the viruses' proliferation.
Gao, Lu,Chen, Keping,Yao, Qin,Chen, Huiqing Korean Society of Sericultural Science 2006 International Journal of Industrial Entomology Vol.13 No.1
The ubiquitin conjugating enzyme 2 (E2) is core component of ubiquitin proteasome pathway (UPP) which represents a selective mechanism for intracellular proteolysis in eukaryotic cells. The E2 has been implicated in the intracellular transfer of ubiquitin to target protein. We show here the involvement of E2 in antiviral immune of Bombyx mori to Bombyx mori nuclear polyhedrosis virus (BmNPV). In this study, mRNA fluorescent differential display PCR (FDD-PCR) was performed with BmNPV highly resistant silkworm strain NB and susceptible silkworm strain 306. At 24 h post BmNPV infection, FDD-PCR with the arbitrary primer AP34 showed that one cDNA band was down-regulated in the midgut of resistant strain, but highly expressed in susceptible strain. The deduced amino acid sequence of this cDNA clone share 99% identity with the recently published B. mori ubiquitin conjugating enzyme E2 (Genbank NO: DQ311351). Fluorescent quantitative PCR corroborated down regulation of E2 in resistant strain. We there conclude that BmNPV infection evokes strong response of susceptible strain including activation of UPP. BmNPV may evolve escape mechanisms that manipulate the UPP in order to persist in the infected host. In addition, the identification of down-regulation of E2 in resistant strain, as well as structure data, are essential to understanding how UPP operates in silkworm antiviral immune to BmNPV disease.
Cloning and Characterization of 6-Phosphogluconolactonase Gene in Silkworm Bombyx mori
Yang, HuaJun,Chen, KePing,Yao, Qin,Guo, ZhongJian Korean Society of Sericultural Science 2007 International Journal of Industrial Entomology Vol.14 No.2
As the genome of B. mori is available in GenBank and the EST database of B. mori is expanding, identification of novel genes of B. mori was conceivable by datamining techniques and bioinformatics tools. In this study, we used the in silico cloning method to get the 6-Phosphogluconolactonase (6PGL) gene of B. mori and analysed with bioinformatics tools. The result was confirmed by RT-PCR and prokaryotic expression. The 6PGL cDNA comtains a 702 bp ORF. The deduced protein has 233 amino acid residues, with the predicted molecular weight of 25946. 72 Da, isoelectric point of 5.41, and contains conserved NagB domains. This gene has been registered in GenBank under the accession number EF198104.